High-throughput assay

ABSTRACT

A homogeneous high throughput assay is described which screens compounds for enzyme inhibition, or receptor or other target binding. Inhibition (or binding) by the library compounds causes a change in the amount of an optically detectable label that is bound to suspendable cells or solid supports. The amounts of label bound to individual cells or solid supports are microscopically determined, and compared with the amount of label that is not bound to individual cells or solid supports. The degree of inhibition or binding is determined using this data. Confocal microscopy, and subsequent data analysis, allow the assay to be carried out without any separation step, and provide for high throughput screening of very small assay volumes using very small amounts of test compound.

FIELD OF THE INVENTION

This invention relates to the high throughput screening of chemicalcompounds for interactions with target molecules.

BACKGROUND OF THE INVENTION

To find lead compounds for drug discovery programs, large numbers ofcompounds are often screened for their activity as enzyme inhibitors orreceptor agonists/antagonists. Large libraries of compounds are neededfor such screening. As a result of developments in this field, it is nowpossible to simultaneously produce combinatorial libraries containinghundreds of thousands of small molecules for screening. With theavailability such libraries, however, has come a need for large scale,rapid screening methods.

For example, the libraries may be contained on microbeads, each compoundbeing present in a picomolar amount. Because the amount of compound isvery small, it is advantageous to conduct the high throughput screeningmethod in very small volumes, e.g., on the order of 1 μl. Such assayscan be performed in the 1536 well plate described in U.S. patentapplication Ser. No. 60/037,636 filed Feb. 18, 1997. Microassays in suchsmall volumes, however, are difficult to accurately and repeatedlyperform using conventional methods.

Receptor binding assays used in high throughput screening typicallyinvolve three steps. First, a labelled ligand is incubated with a targetreceptor in the presence of compound to be tested for inhibition ofligand/receptor binding. Second, the receptor and ligand (and compound)are separated using filtration and/or washing of an immobilizedreceptor. Finally, the amount of labelled ligand bound to the receptoris quantified. This conventional screening is a `separations-mode`assay, i.e., one in which the bound ligand is physically separated fromthe free ligand using either a filtration membrane or the selectiveadhesion of either bound or free component to a surface (e.g., thesurface of a microtiter plate).

Separation, however, is time-consuming and therefore slows highthroughput screening. It can also, if fluid handling steps employed arenot sufficiently precise, create variations in the signal generated inthe assay and can disturb equilibrium binding conditions. Furthermore,separation is difficult to automate and is potentially hazardous whenradioactive materials are involved. These problems are particularlyacute in assays conducted in microvolumes using small amounts of testcompound.

It is therefore advantageous in high throughput screening to distinguishbound and free ligands in a homogeneous assay, i.e., one that eliminatesthe need for separation. To be particularly useful in screening largescale combinatorial libraries, such an assay should readily permit smallvolumes, and small amounts of test compounds, to be used.

A homogeneous assay is described in U.S. Pat. No. 4,568,649 whichemploys beads that are impregnated with a scintillant (these arecommercially sold as Scintillation Proximity Assay beads (SPA™, AmershamCorp., Arlington Heights, Ill.)). The beads are also coated with aligand that is capable of binding with radio-labelled target in asample. When the ligand binds to the radio-labelled target, thescintillant on the bead is activated by the radiolabel. The level oflight energy produced by the scintillant indicates the amount of boundlabelled target in the sample. This method, however, requires handlingof radioactive reagents and is somewhat limited in sensitivity.

Another homogeneous assay is known in which signal is generated whenlabeled ligand and labeled target interact. One label is an energydonating Eu-cryptate having a long-lived fluorescent excited state andthe other is an energy-accepting protein, allophycocyanin, having ashort fluorescent excited state. Energy transfer occurs between thelabels when they are less than 7 nm apart. During the assay, theEu-cryptate is excited by a pulsed laser, and its fluorescent emissioncontinually re-excites the allophycocyanin, whose fluorescence ismeasured by a time resolved fluorescence reader. This method, however,requires labeling of both the ligand and the target and is not assensitive as some other commercially available assays. Also,allophycocyanin is a very large, multimeric protein which can affect theassay in an unpredictable manner.

A fluorescent imaging plate reader has been used to perform opticalscreening in cell-based kinetic assays that measure membrane potentialand intracellular calcium. The assay employs an optical method thatlimits the depth of field measured by a CCD camera to the bottom of anassay well, where fluorescence in a layer of live adherent cells ismeasured. By limiting the depth of field to the cell layer, backgroundfluorescence from extracellular dye is reduced. Data is obtained overtime measuring, e.g., depolarization of cells (Schroeder et al., (1996)Journal of Biomolecular Screening 1:75-80). This method, however, useslive cells that require maintenance for the period of the assay,necessitating complicated integrated fluid handling to trigger rapidcellular events. Such handling is very difficult, if not impossible, toperform in the microvolumes that are used in high throughput screeningof small amounts of library compounds. Also, measurement is taken of abulk sample, i.e., of the entire layer of cells and the assay does notdiscriminate between fluorescence bound to individual suspended cellsand background fluorescence.

It is therefore an object of the present invention to provide an assayfor high throughput screening that does not require a separation step,i.e., is homogeneous.

It is another object of the invention to avoid radioactive waste, and toavoid labeling of both ligand and target molecule.

It is another object of the invention to provide an assay which isreadily adaptable for miniaturization in microvolumes, and which ishighly sensitive.

It is another object of the invention to provide a high throughputscreening method for detecting activity of small amounts of compounds,such as are found in combinatorial libraries of beads having picomolaramounts of compound thereon.

SUMMARY OF THE INVENTION

The present invention relates to a high throughput assay for rapidlyscreening a plurality of compounds. The assay determines the degree ofinhibition by the compounds of a ligand/receptor interaction, or of anenzyme catalyzed reaction, or the degree of binding of library compoundsto a target molecule. Inhibition (or binding) by the library compoundscauses a change in the amount of an optically detectable label that isbound either to suspendable cells or to suspendable solid supports. Thedegree of inhibition (or binding) is determined by measuring, bymicroscopy, the amounts of label that are bound to individual cells orsolid supports. These amounts are compared with the amount of label thatis not bound to individual cells or solid supports (i.e., backgroundsignal). The degree of inhibition or binding is determined using thisdata. Preferably, measurement is performed using a confocal microscope.The assay is homogeneous, i.e., no separation step is required to removeunbound label, since the amount of bound label is distinguished byscanning of the individual cells or solid supports. The method allowsexceptional sensitivity and high throughput to be obtained in assaysusing small volumes, and small amounts of test compound.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1D are schematic depictions of an embodiment of the presentinvention employing a suspension of cells having surface receptors thatbind to fluorescently labelled ligand. Active library compounds inhibitcell-bound fluorescence, causing a decrease in signal that is measuredfor individual cells.

FIG. 2 is a schematic depiction of an embodiment of the presentinvention employing beads having target molecules coated thereon thatbind to fluorescently labelled ligand in the assay medium unless anactive library compound is present.

FIG. 3 is a graph showing the mean fluorescent intensity (MFI)calculated for binding of Cy5-NKA to NK2R/CHO cells.

FIG. 4 is a graph showing fluorescence associated with cells calculatedas percent of control for displacement of Cy5-NKA by SR48.968.

FIG. 5 is a graph of F-values obtained for displacement of fluorescentlylabelled IL-8 by unlabeled IL-8.

DETAILED DESCRIPTION OF THE INVENTION

All patent applications, publications, or other references that arelisted herein are hereby incorporated by reference.

The present invention allows high throughput screening of compounds,such as those found in combinatorial libraries, to determine active drugcandidates. The method allows substantial reduction in the time requiredfor screening such libraries over that required by non-heterogeneousmethods. It also eliminates the need for disposal of washed reactantsand is highly adaptable to performance in micro-volume assay vessels. Inaddition, the method improves over conventional homogeneous assaymethods in its higher signal to noise ratio and in requiring use of onlyone label. It provides a sensitive assay that allows successfulscreening of very small amounts of compounds derived from microbeadlibraries, thereby allowing more assays using those microbeads, andfaster, more efficient screening.

The method of the invention involves measurement of the amounts of boundand free signal in the assay by microscopy. This can be carried out, forexample, by sequentially viewing different depths in the sample using aconventional microscope employing a narrow depth of focus. According tothe preferred method of the invention, however, confocal microscopy isemployed to individually determine the amount of bound signal toindividual cell sized particles.

Confocal microscopy confines detection of an illuminated object, orsample, to a thin object plane. A view of a "slice" of the object, orsample, is obtained. This is achieved, for example, by placing a spacialfilter, such as a pinhole, in the image plane located between theobjective lens and a detector. Only light emitted from a narrow regionnear the object plane converges through the spacial filter. Light fromother planes is blocked by the filter. Images are obtained of the objectplane, e.g., by scanning, in sequence, the points in the field of view,to obtain the "slice".

Confocal microscopy using laser scanning is particularly preferred foruse in the invention. A suitable laser scanning microscope is sold as"IMAGN/2000" by Biometric Imaging Inc. (Mountain View, Calif.). Laserscanning microscopes are also described in U.S. Pat. Nos. 5,556,764 and5,547,849. These microscopes are conventionally used to analyze bloodwithin a capillary tube to determine the number of cells labelled byfluorescent antibodies.

Non-laser scanning confocal microscopes are well known and can also beused to practice the invention. For example, confocal microscopes usingspinning Nipkow disks, or similar arrangements can be used, if desired.Such microscopes are described, e.g., in Dixon (1996) Nature, 383:760;Juskaitis et al. (1996) Nature 383:804; Petran et al. (1968) J. Opt.Soc. Am. 58, 661; and Xiao et al. (1988) Appl Phys. Lett. 53:716.

Useful confocal microscopes are also described, for example, in U.S.Pat. Nos. 5,032,720; 5,120,953; 5,260,578; 5,304,810; 5,283,684;5,351,152, and 5,162,946.

According to the method of the invention, data obtained by confocalmicroscopy are analyzed to determine the difference between signalassociated with individual suspended cells or solid supports, andbackground signal, and to obtain a measure of inhibition or binding.Conventional confocal microscopy for, e.g., counting CD4+ cells, is notconcerned with this determination, or with high throughput screening ofcompounds.

Any desired optically detectable label can be used in the presentinvention, including fluorescent labels and chemiluminescent labels. Ifthe label is chemiluminescent, it is preferred that it generate ashort-lived signal. Enzymes that produce a visible color change in thepresence of appropriate substrate, such as horse radish peroxidase andalkaline phosphatase, can also be used. Fluorescent labels are referredand are described below with respect to preferred embodiments of theinvention. Other optically detectable labels, however, may besubstituted in these embodiments if desired.

In one embodiment, the assay screens compounds for inhibition of bindingof a fluorescently labeled ligand to a target molecule contained on thesurface of either suspendable cells or solid supports. The degree ofbinding inhibition is determined by measuring, through confocalmicroscopy, amounts of labeled ligand bound to individual cells orsupports in the presence of the compounds.

In this embodiment, a suspended cell-membrane bound receptor can becontacted with fluorescently labelled ligand, in the presence of librarycompounds to be screened for inhibition of receptor binding. (The term"receptor" is used herein to encompass receptor domains as well as wholereceptors.) As an example, a library of compounds to be screened forinhibition of the binding of IL-8 to its cell surface receptor iscontacted with a suspension of cells bearing the receptor in thepresence of fluorescently labelled IL-8. When examined by confocalmicroscopy, cells bound to labeled IL-8 appear as regions of increasedfluorescence on a background of relatively constant "free" label. Theamount of cell associated fluorescence is less in an assay where activecompound inhibits binding of ligand to receptor.

This embodiment is schematically depicted in FIG. 1. FIGS. 1A and 1Cshow a cell with attached receptors (`Y`), as they bind tofluorescently-labelled ligands (solid square flagged with `F`). In FIG.1C, fluorescently-labelled ligands are displaced by library compound(open triangles). FIGS. 1B and 1D schematically indicate fluorescencecollected from sections of samples with and without active librarycompounds, respectively. Presence of a "spot" relative to backgroundfluorescence indicates bound ligand, while the background fluorescenceitself results from free ligand. Confocal microscopy allows measurementof individual cell-sized "spots" of bound fluorescence. The amount offree fluorescent ligand can be simultaneously determined. In FIG. 1B,the cells are significantly more fluorescent than the background andtherefore show up as more intense spots. In FIG. 1D, the effect ofdisplacement by an active library compound is illustrated. The samecells have become dimmer or indistinguishable from background as aconsequence of ligand displacement. Thus, the loss of `bound` signalindicates an active molecule in a high-throughput screening assay. Sincereceptor binding assays are typically conducted with an excess ofligand, the amount of background fluorescence does not normally change.

In the assay of the invention, the amount of fluorescence associatedwith individual cells (or solid supports) in the assay is totaled. Thistotal provides a measure of the amount of binding between ligand andtarget. This amount is compared to the amount of free fluorescence toarrive at a value indicative of the activity of the library compound(the amount may increase or decrease for active drug candidates,depending on the way in which the assay is set up). Specificmethodologies for arriving at particular values indicating binding (orinhibition of binding) are described below. While backgroundfluorescence can be individually measured in each assay, this may not benecessary where the background fluorescence is relatively constant.

The method of the invention is particularly advantageous in increasingthe level of signal to background noise. By eliminating signal fromlabel contained in solution outside of the "slice" containing themeasured particles or cells, the background noise is significantlyreduced. The ratio of signal to background noise was found to be aboutfifteen times lower in a commercially available conventionalheterogeneous (i.e., separation-based) assay using ¹²⁵ I labeled ligandthan in the method of the invention.

The method is especially effective in measuring fluorescence for samplesthat have been allowed to settle. Confocal microscopy allows accuratemeasurement of a "slice" or "section" of liquid in a container. Thus,measurement can be taken of, e.g., the bottom 10% of the sample wherefluorescence bound to the cells or solid particles is concentrated. Thisis not possible in prior art assays using conventional optical detectionsince such assays do not eliminate signal from the volume above thesettled cells or solid particles. Elimination of this signal accounts,in part, for the very high signal to noise ratio achieved by the methodof the invention.

Preferably, the suspended cells (or suspended solid supports) areallowed to settle for about 10 minutes or more, so that more than about75% of the cells or supports are contained in less than about 25% of thevolume of the assay container, i.e., a cell or solid support layer formson the bottom. Most preferably, more than 90% of the cells or supportsare allowed to settle in less than about 10% of the volume of thecontainer. In one preferred embodiment, the thickness of the layer ofcells or supports is about the same as the thickness of the confocalobject plane. The time required for settling is a function of columnheight, and so is higher, e.g., for samples in 96 well plates than forsamples in 1536 well plates.

Any desired combination of ligand and receptor can be employed in theassay of the invention to test for active inhibitors. Non-limitingexamples of ligands and their cell based receptors include neurokininand NK2R cells, and IL-8 and IL-8B/CHO cells. These receptors andligands are discussed further in the Examples below.

Other examples of ligands and receptors include, but are not limited to,insulin/insulin receptor, bradykinin/bradykinin receptor,erythropoietin/EPO receptor, and leptin/Ob receptor. Cell lines forperforming these assays are available. For example, IM-9 (ATCC CCL-159)constitutively expresses human insulin receptor. IMR-90 (ATCC CRL 7931)constitutively expresses bradykinin B2 receptor and can be stimulatedwith interleukin-1δ to produce bradykinin B1 receptor. Cells thatexpress EPO receptor are described by Kitamura et al., J. CellularPhysiology 140:323-334 (1989). Cells expressing leptin receptor (i.e.,OB receptor) are described by Tartaglia et al., Cell 83:1263-1271(1995).

In another embodiment of the invention, a target molecule is bound, e.g.via a biotin/avidin association, to suspendable solid supports, andlibrary compound and fluorescently labeled ligand in solution arecontacted with the supports. Active compound causes a decrease insupport-associated fluorescence by displacing fluorescent ligand fromthe target, and the presence and/or potency of the test compound isquantitated. The brightness of fluorescence of the supports diminishesin proportion to the potency of the test compound. This method isschematically depicted in FIG. 2.

"Suspendable solid support" is intended to refer to any solid supportcapable of being suspended in a liquid. The support should be smallenough so it does not block optical access to the rest of the solutionupon settling to the bottom of the assay well. On the other hand, thesupport should be large enough so that it does not remain in suspensionfor an extended period of time after the assay components are combined.The preferred supports are less than about 50 μm in diameter, mostpreferably less than 10 μm in diameter. The diameter of the supports ispreferably less than, although not significantly less than, thethickness of the confocal object plane. The supports are preferablygreater than 1 μm in diameter, so that the suspension does not requirecentrifugation to condense the supports to the bottom of the assaycontainer.

A preferred suspendable support is a 6.2 μm bead made of polystyrene andcommercially available from Spherotech (Libertyville, Ill.). Such beadsare avidin coated, typically containing 10⁶ binding sites per bead. Anysuitable suspendable solid support, however, can be employed, includingcellulose beads, controlled pore-glass beads, silica gels, and othertypes of polystyrene beads (optionally cross-linked with divinylbenzeneand optionally grafted with polyethylene glycol and optionallyfunctionalized with amino, hydroxy, carboxyl, or halo groups).Additional supports include grafted co-poly beads, poly-acrylamidebeads, latex beads, dimethylacrylamide beads (optionally cross-linkedwith N,N¹ -bis-acryloyl ethylene diamine), glass particles coated withhydrophobic polymers, etc., (i.e., having a rigid or semi-rigidsurface). Divinylbenzene-crosslinked, polyethyleneglycol-graftedpolystyrene type beads can be used, such as TentaGel S-NH₂ ® beads (RappPolymere, Tubingen, Germany).

The solid support can be coated with any desired target, including, butnot limited to, hydrolases (including proteases, esterases, nucleases),ligases (DNA or RNA based) and transpeptidases, as well as bindingproteins such as antibodies, and DNA-binding proteins, and domains ofthose proteins.

The target (or ligand) coated on the solid support may be bound theretoby any desired means. It may, for example, be biotinylated and thennon-covalently linked to a streptavidin coated support. It is alsopossible to bind the target (or ligand) to antibodies (which arespecific for the target) that have been coated on the support. Covalentlinkages are also known in the art.

The support may also be coated with a recombinantly produced receptor,or receptor binding domain. This is particularly advantageous forreceptors or domains that are not normally expressed on the cellsurface. For example, nuclear receptors, such as steroid receptors, areadvantageously expressed recombinantly, and employed in the microbeadassay of the invention. One such receptor is human recombinant estrogenreceptor (Alexis Biochemicals, San Diego, Calif.). For a cell surfacereceptor, however, it is preferred to use suspended cells expressing thereceptor as opposed to beads having the receptor bound thereto.

It is also possible, for example, to coat the suspendable solid supportswith ligand, and perform the assay of the invention with labelledreceptor in solution, in the presence of compounds to be screened forinhibition of ligand/receptor binding.

It is also possible, according to the invention, to incubatefluorescently labelled library compounds in solution with suspendedcells or solid supports, and measure the binding between said compoundsand cells or supports in the absence of ligand. In other words, theassay provides a direct measure of binding between the compounds andtarget molecule on the cells or supports, without the need to add aligand that is displaced by the compounds.

In another embodiment, a library of compounds is assayed for inhibitionof an enzyme catalyzed reaction and the amounts of fluorescence bound toindividual suspendable solid supports measured to determine the degreeof inhibition. For example, in one such assay, the amount offluorescence bound to a microbead in the presence of inhibitorycompounds is greater than for non-inhibitory compounds. The amounts offluorescence bound to individual beads are determined by confocalmicroscopy. Using this type of assay, inhibition can be determined of aprotease, such as cathepsin D, which cleaves fluorescently labelledsubstrate bound to the solid support. For cathepsin D, the substrate canbe a peptide, e.g. lys-pro-ile-glu-phe-phe-arg-leu, linked at one end tothe microbead and at the other end to the fluorescent label Cy-5; eitherlinkage can be accomplished using a spacer such as gamma aminobutyricacid.

It is also possible, using this type of assay, to determine inhibitionof endonuclease cleavage of fluorescently labelled oligonucleotide. Theendonuclease is placed in solution with library compound and suspendablesolid supports that are coated with fluorescently labelledoligonucleotide substrate. Upon cleavage of the substrate, fluorescentlylabeled product is released from the supports. The amount offluorescence that remains bound to the bead increases where activeinhibitor is present.

In another assay for enzyme inhibition, both enzyme and fluorescentlylabelled substrate are incubated in solution with test compound andmicrobeads coated with a ligand. The ligand (such as an antibody)specifically binds to the reaction product of the enzyme catalyzedreaction, the reaction product retaining the fluorescent label. Forexample, inhibitors of tyrosine kinase can be determined in an assay inwhich kinase and fluorescently labelled peptide substrate are insolution. The peptide substrate contains a tyrosine amino acid in themiddle of its sequence, and the reaction product containsphosphotyrosine. The assay solution contains suspendable microbeadscoated with antibody for the phosphotyrosine containing reactionproduct. Successful inhibition by a library compound results in adecrease in fluorescence bound to beads as compared with controls.

In assays of the invention in which inhibition of an enzyme catalyzedreaction is determined, the inhibitory compounds can inhibit by anymechanism. For example, they can inhibit by binding to enzyme, bindingto substrate, binding to a complex of enzyme and substrate, or bindingto a complex of enzyme and product.

Preferably, the assay of the invention is performed using a microtiterplate having microvolume containers, such as the 1536 well platedescribed in U.S. patent application Ser. No. 60/037,636 filed Feb. 18,1997. A confocal scanning microscope sequentially scans the bottom ofeach well in the microtiter plate.

The method can also be carried out in conventional 96 well microtiterplates, or in any other container or on any surface capable of holdingliquid samples and of being scanned by a confocal microscope. Examplesinclude 12-well, 24-well, 384-well, 864-well plates, andmicroscope-slides.

In the embodiment of the invention in which receptor-bearing cells areemployed, the desired density of cells will preferably be between about100 and 1000 cells per microliter in a 1536 well plate and between about30 to 300 cells per microliter in a conventional 96 well plate.Typically, it is believed necessary to measure signals from at least 100to 1000 cells per sample to obtain a statistically relevant result. Theoptimum density can be determined using these concentrations as aguideline, as well as the size of the particular cells employed, and bymeasuring the signal provided in assays using known inhibitors of theligand/receptor interaction. The area scanned can be limited to reducescanning time and thereby increase throughput, as long as the number ofcells measured is sufficient.

If suspendable supports are employed in the preferred size of about 6μm, the preferred density of supports will generally be in the samepreferred range as for cells. The density varies depending on the sizeof the supports and the amount of target affixed to each support.

The signal detected according to the invention, is preferably generatedby a fluorescent label. The label can be attached to a ligand whichbinds to a receptor or other target molecule. It is also possible to uselabelled receptor (or other target molecule) in certain embodiments ofthe invention. Also, if desired, a "secondary labelling" approach can beused in which labelled antibody probes, e.g., for unlabelled ligand,receptor, or target molecule.

It is also possible to conduct an assay according to the invention inwhich library compounds themselves are fluorescently labelled. Forexample, a library of compounds that are primary amines can be labelledwith an amine-specific fluorescent label (e.g., monofunctional Cy5-NHSester). The compounds can then be tested for direct binding to a targetmolecule on a cell or suspended support. The amount of boundfluorescence correlates to the degree of binding.

Fluorescent labels suitable for use in the invention are well known andinclude cyanine dyes such as Cy-5, Cy-5.5, and Cy7 (Amersham Corp.),fluorescein, rhodamine and Texas red. In the embodiment of the inventionemploying cells, it is preferred that the fluorescent label fluoresce ata relatively high wavelength, i.e., higher than about 450 nm, to avoidinterference from cell originating fluorescence and fluorescenceoriginating from glass and plastic containers. The labels mostpreferably fluoresce above 600 nm, and at less than about 800 mn. Labelsthat excite at about 400 nm can avoid photobleaching caused by near-UVlight.

Non-fluorescent labels can also be used in embodiments of the inventiondescribed above. In one embodiment using the chemiluminescencegenerating label luciferase, a receptor is coated on suspendable solidsupports. The coated supports are incubated with luciferase conjugatedligand, luciferase substrate, and compound to be tested. In the absenceof inhibition of receptor/ligand binding, the luciferase becomesassociated with the suspendable solid supports, and the chemiluminescentsignal that results from luciferase's enzymatic action concentratesaround the supports. In the presence of inhibitory compounds, the signalassociated with the supports decreases.

In a separate embodiment of the invention, the assay is performed todetermine the degree of binding to a treated surface of an insuspendablesolid support. The support can be a container, or vessel, itself, suchas the bottom of a microtiter plate. Alternately, the insuspendablesupport can be, e.g., a disc. In the embodiment in which the support isthe bottom of the well of the microtiter plate, the plate is coated witha target molecule, and then exposed to labelled ligand. A confocalsection including the bottom layer of the plate is measured foroptically detectable signal. Free signal is measured in other confocalsections that do not include the bottom of a microtiter plate. Thesignal bound to the support can then be calculated. This method isadvantageous in that scanning can be rapidly performed. Individualcells/beads do not need to be identified, resulting in higherthroughput. Use of a thin confocal object plane is preferred to excludesignal emanating from above the coated plate, and to maintain a highsignal to noise ratio. In one embodiment of this method, the confocalobject plane is less than about 10 μm.

It is preferred that the compounds assayed in the high throughput methodof the invention be derived from combinatorial libraries on polymerbeads. By synthesizing sufficient compound on each bead for a fewassays, compound handling is reduced or eliminated. Such beads, e.g.,can contain on the order of 100 picomoles of compound per bead, andtests are often performed at concentrations of about 1 μM. With suchbeads, a test volume of 1 μl is advantageous since it is possible to useone bead for up to about one hundred tests.

Preferably, the library compounds are eluted from the beads andevaporated to dryness in microtiter plates in preparation for the assay.Compounds on beads can be released by photocleavage, or another type ofcleavage. Cleavage of photocleavable linkers is preferred. Such linkers,and methods for their cleavage, are described in Barany et al. (1985) J.Am. Chem. Soc. 107:4936. Examples of other linkers and the relevantcleavage reagents are described in WO 94/08051.

Using combinatorial libraries prepared on beads, the identity of activecompounds is preferably determined using the encoding system describedin WO 94/08051, and in U.S. patent applications Ser. Nos. 08/436,120 and08/239,302 (which correspond to WO 95/30642). In this system, chemicaltags encoding the identities of the compounds are applied to the solidsupports. The identity of the compound on a given support can bedetermined by detaching the chemical tags from the support, identifyingthe tags by, e.g., gas chromatography, and correlating the identities oftags with the identity of the compound. Once an active compound isidentified, the corresponding bead (which had contained the compound)can be examined, and the identity of the compound determined byreleasing the tags and decoding by this method.

When several large libraries are available for testing, it may beadvantageous to "scout" each library by placing more than one testcompound in each assay container. Assay containers having an activecompound can be further investigated by individually evaluating each ofthe plurality of compounds present in such containers. Screening at"high density" in this manner allows one to statistically evaluate thenumber and potency of active compounds in each library. Libraries whichcontain the most active compounds can be more thoroughly tested. If theproportion of active compounds screened in the assay is high, a secondassay of the active compounds may be performed at lower concentrationsto select only the most active compounds to choose those that should befurther evaluated.

The invention is illustrated by the following examples, which are notintended to limit the scope of the invention.

EXAMPLE 1 Screening of Compounds for Receptor Binding

The invention was demonstrated using confocal microscopy apparatus andsoftware originally intended for fluorescent cytometry. The dataobtained were further analyzed to provide values indicative of theamount of binding between ligand and a cell surface receptor.

1) Synthesis of Ligands

(a) Cy5-labelled Neurokinin-A (NKA).

Neurokinin-A (HKTDSFVGLM) was purchased from Cambridge ResearchBiochemicals (PP-05-0826A), and monofunctional Cy5 dye was purchasedfrom Amersham (as the Fluorolink™ conjugation kit, cat. # A25001). NKA(1 mg) was dissolved in 0.88 mL bicarbonate buffer (100 mM NaHCO₃, pH9.3), and Cy5 dye (about 1 mg) was dissolved in 0.13 mL bicarbonatebuffer. The two solutions were mixed and incubated for 1.5 hr at roomtemperature, then transferred to 5° C. for an additional 17 hrs. Theconjugate was purified by HPLC (gradient 20-40% CH₃ CN in H₂ O, 0.1%TFA, on a Vydac® analytical C18 column, R_(f) 8.9 min @ 1.5 mL/min) toyield 157 mol (18% theoretical yield). Identity of the ligand wasverified by competition vs. receptor and mass spectrometry.

(b) Cy5-labelled IL-8.

Human interleukin-8, Ser72→Cys mutant (hIL-8(S72C)) was cloned fromhuman cDNA using PCR techniques and sequenced to confirm the mutationand the sequence. Monofunctional Cy5-iodoacetamide was obtained fromAmersham. To 500 nM Cy5-iodoacetamide was added 200 nmol (200 μl of a 1mM stock) of hIL-8(S72C) in 20 mM sodium phosphate, pH 6.5, 400 mM NaCl.The tube was vortexed and placed in the dark at ambient temperature.HPLC analysis indicated that after 48 hours the reaction was complete.The product was purified (2×100 μl injections) from unreactedCy5-iodoacetamide and oxidized hIL-8(S72C) by HPLC. Sample wasreconstituted in 50 mM sodium phosphate, pH 7.2 at an estimatedconcentration of 400 μM. A more accurate concentration was thendetermined from a fluorescence spectrum of an aliquot of this sample.Further characterization indicated that hIL(S72C)-Cy5 had a mobilitysimilar to wild type IL-8 on a 16% SDS-PAGE analysis and displayed aK_(i) of 2 nM in a conventional ¹²⁵ I!IL-8 ligand displacement assay.

2) Cell lines

(a) Cells expressing neurokinin-2 receptor (NK2R/CHO) were obtained.Cells expressing NK2R are well known, and readily obtained by thoseskilled in the art. For example, NK2R expressing cells are described inthe following references: Arkinstall, S., M. Edgerton, et al. (1995)."Co-expression of the neurokinin NK2 receptor and G-protein componentsin the fission yeast Schizosaccharomyces pombe." FEBS Lett 375(3):183-7; Bradshaw, C. G., K. Ceszkowski, et al. (1994). "Synthesis andcharacterization of selective fluorescent ligands for the neurokinin NK2receptor." J Med Chem 37(13): 1991-5; Grisshammer, R., J. Little, et al.(1994). "Expression of rat NK-2 (neurokinin A) receptor in E. coli."Receptors Channels 2(4): 295-302; Lundstrom, K., A. Mills, et al.(1995). "High-level expression of G protein-coupled receptors with theaid of the Semliki Forest virus expression system." J Recept SignalTransduct Res 15(1-4): 617-30; and Turcatti, G., K. Nemeth, et al.(1996). "Probing the structure and function of the tachykininneurokinin-2 receptor through biosynthetic incorporation of fluorescentamino acids at specific sites." J Biol Chem 271(33): 19991-8.

b) Cells expressing IL-8A and IL-8B receptor (IL-8A/CHO and IL-:8B/CHO)were obtained as follows:

CHO IL-8A and IL-8B cell lines were prepared by cationic lipopolyamine(lipofectamine, GIBCO BRL) mediated transfection of CHO-K1 cells (ATCC)with pCDNAIII plasmids encoding the sequences of human IL-8A or IL-8B.Cells were cultured under G418 selection (1 mg/ml) in DMEM, 10% fetalbovine serum, 2 mM L-glutamine, and 2% non-essential amino acids andclonal lines expressing the highest receptor levels were maintained foruse in these experiments. The human IL-8A receptor cDNA was cloned fromHL-60 cell (ATCC) mRNA. (Clones encoding IL-8A are described in Ahuja etal., (1992) Nature Genetics 2:31.) First-strand cDNA was synthesizedusing M-MLV reverse transcriptase (Promega Riboclone™ cDNA synthesissystem) and IL-8A cDNA was amplified by polymerase chain reaction usingprimers, 5'CCGAATTCGACATGTCAAATATTACAGATCC3' and5'GCTCTAGATCAGAGGTTGGAAGAGAC3'. The PCR product was digested withEcoRI+Xbal and ligated into EcoRI/Xbal/calf intestinalphosphatase-digested pcDNA3 vector (Invitrogen). The DNA sequence of onecandidate was confirmed using the Promega Silver Sequence™ method. Togenerate the human IL-8B expression clone, an approximately 1.8 kb cDNAfragment was recloned from pBluescript clone BS-p3 (Murphy and Tiffany,(1991) Science, 253:1280) into the pcDNA3 vector, using EcoRI and Xhol.

3) Assay

(a) Binding of Cy5-NKA to NK2R/CHO cells

A culture of NK2R/CHO cells, near confluence, was washed with 12 mLDPBS(Mg⁺² & Ca⁺² free), trypsinized by adding trypsin (2 mL/T-25 flask),incubating at 37° C. for about 5 min, then quenching with 10 mL media.Cells were then counted, and diluted to the desired final concentrationof cells (5,000 cells/25 μL). The assay was set up with the followingcomponents: Buffer (1× BSS+0.2% BSA, containing thiorphan andbacitracin) containing varying concentrations of Cy5-NKA (0.76 to 136 nMfinal) 10 μL; Cells, 40 μL. The plate was covered and wrapped inaluminum foil to protect from light and left to shake for 1 hr at roomtemperature. For each sample, 25 μL was removed, placed in an IMAGN2000™ capillary, and read using IMAGN™ software (Biometric Imaging,Inc.) The sample was allowed to settle until the cells rested on thebottom of the well. The time require for settling was typically 10minutes.

(b) Displacement of Cy5-NKA bound to NK2R/CHO cells by SR48,968.

SR-48,968 is a known inhibitor of the binding of NKA neurokinin 2receptor. Setup and assay were performed as in (a), at a fixedconcentration of Cy5-NKA of 5 μM, and diluting stock concentration ofSR-48,968 (stock 1 mM, 1.6% DMSO, in BSS) from 88 nM to 8.8 pM inCy5-NKA-containing buffer.

(c) Displacement of Cy5-IL-8 bound to cells by unlabelled IL-8.

A culture of IL-8B/CHO cells, near confluence, was trypsinized by addingtrypsin (2 mL/T-25 flask), incubating at 37° C. for about 1 min, thenquenching with 4 mL media. Cells were then counted, and diluted to thedesired final concentration (15,000 cells/25 μL). The assay was set upin a 96-well microliter plate (100 μL total volume), consisting of thefollowing components: Buffer (1× BSS) 30 μL; Cells, 25 μL; Backgroundfluor (Tris-Cy5, 60 μM), 25 μL; hIL-8(S72C)-Cy5(20 nM), 10μL; unlabelledIL-8 (various concentrations, diluted in BSS), 10 μL. The plate wascovered and wrapped in aluminum foil to protect from light and left toshake for 1 hr at room temperature. For each sample, 85 μL was removedand placed in an IMAGN 2000™ capillary, and read using IMAGN software.

4) Data Analysis

The data that results from the IMAGN system consists of a tab delimitedtext file that contains information about each cell that has beenidentified in the field. The IMAGN system provides position, shape, andintensity data (for two channels, Cy5 and Cy5.5) for each cell (orcell-sized object) in the imaging field, as well as baselineinformation. (In a typical analysis using this type of equipment, cellsare identified by two or more contiguous pixels having intensitiessignificantly greater than the baseline signal.) Statistical parameters(e.g., standard deviations) of the data are also tabulated. This datacan be analyzed to give a scalar value for the sample which provides ameasure of the amount of binding. Three possible ways to perform thisfurther analysis are described below.

(a) Mean Fluorescence Intensity.

This value is derived from the data table as the mean value across allcells of "MaMO". Channel 0 (zero) is the Cy5 fluorescence channel, andthe MaMO value is calculated as the peak Cy5 fluorescence (correspondingto fluorescence bound to the cell) with the minimum (baselinefluorescence) value subtracted out. "MaMO" is tabulated for each cell inthe field.

This analysis is particularly useful for assays that involve high fluorconcentrations and relatively high levels of occupation of cell surfacereceptors. It may not be advantageous where all cells cannot bedetected, e.g., at low fluor concentrations or low levels of occupationof cell surface receptors. Analysis of F value (explained below) canprovide more accurate data under these circumstances. Specifically, moreweakly fluorescent cells (by virtue of their size and/or idiosyncraticbinding characteristics) will `disappear` first in the imaging fieldwhen being displaced by a competing ligand. ("Disappearance", in thissense, means that the pixels comprising the cell's image are notsignificantly higher in intensity than the baseline image.) Two changesare observed experimentally when challenging a fixed fluorescent ligandconcentration with a competitor. First, the overall intensity of thecells decreases (i.e., the average MaMO value decreases). Second, thenumber of observable cells decreases, since the weaker cells vanish intothe background. The F-value measures the total fluorescence in the celllayer and therefore provides a more accurate measurement when all cellscannot be counted.

(b) F-value.

The F-value is determined by multiplying the MFI by the total number ofcells loaded into the well (as opposed to the number of fluorescentcells counted), and dividing by 1000. This value provides the totalfluorescence of the cell layer, and includes cells whose fluorescence isso weak as to be undetectable in a given scan by the confocalmicroscope. The F-value is believed to generally provide a more accuratemeasure of binding in a given sample.

(c) Percent of Control.

Percent of control analysis can be performed using values obtained fromeither an MFI or F-value analysis, and results in normalization of thisdata. It is calculated according to the following formula for therelevant value. "Max" and "min" refer to the maximum and minimumfluorescence for the relevant value.

    % Control=((Value-Min)/(Max-Min))×100

The MFI calculated for binding of Cy5-NKA to NK2R/CHO cells is shown inFIG. 3. These results show a maximum MFI of 7,720 and a Kd (dissociationconstant) of 3.9 nM.

Results calculated as a percent of control for displacement of Cy5-NKAby SR48.968 are shown in FIG. 4. An IC₅₀ of 0.575 nM was determined andan estimated Ki of 0.29 nM. An R² value of 0.963 was determined,indicating "goodness of fit" of the experimental data to thetheoretically determined values.

The Hill coefficient measured was 0.996. The Hill coefficient is ameasure of the cooperativity in binding. For a simple system, where onereceptor binds to one ligand, the Hill coefficient is 1. For polyvalentsystems, the Hill coefficient can vary from one, but ideally is a smallinteger for polyvalent ligands, or the reciprocal of a small integer forpolyvalent receptors. The NKA/NK2R and IL-8/IL-8BR systems involvemonovalent receptors and monovalent ligands.

Results, calculated as F-values, for displacement of labelled IL-8 byunlabelled IL-8, are shown in FIG. 5. The graph insert provides theequation for a four-parameter curve fit:

    y=((M1-M4)/(1+(M0/M3).sup.M2))+M4.

where M is the maximum value, M4 is the minimum value, M3 is themidpoint of the curve (the apparent Kd), and M2 is the slope at themidpoint.

Binding constants for the above results can be determined, by Scatchardanalysis, which involves the measurement of bound ligand as a functionof free ligand concentration. Specifically, the ratio of bound to freeligand is plotted versus the bound ligand concentration. A straight lineresults, whose slope is the negative reciprocal of the dissociationconstant for the ligand, and whose x-intercept is the maximum bindingvalue (B_(max)).

The results of these experiments demonstrate that the method of theinvention allows precise quantitative analysis of ligand/target binding,and can be used for a variety of targets commonly employed incombinatorial library screening. The method also allows rapid detectionand analysis in high-throughput screening. The variablity in the resultsis low and the results strongly correlate with those obtained byconventional methods of analysis. The method is easily performed in amicroscopic volume. It is therefore well-suited for high-throughputscreening in microliter samples, such as when using a 1536-well plate,and allows efficient use of small amounts of library compounds derivedfrom coated microbeads.

5) Comparison with conventional assay.

In receptor binding assays, the sensitivity of the assay is limited bybackground. It is possible to compare sensitivities of different assaysby calculating the ratios of maximum signal to background. For the assaydescribed above involving IL-8 binding, the assay of the invention wasfound to provide a signal (F-value) of 6300 on a non-specific backgroundof 200, for a signal-to-background ratio of 31.5. For example, ¹²⁵I!IL-8 binding analysis using a similar concentration of labelled IL-8results in a signal of 85,000 cpm on a non-specific background of 39,000cpm, for a signal-to-background ratio of 2.1. Based on these values, theimprovement in sensitivity is about 15-fold using the assay of theinvention.

EXAMPLE 2 Screening of compounds for SH2 domain binding

A high-throughout assay is performed to determine inhibitors of bindingof ligand to the SH2-domain of human Grb2. Grb2 is an adaptor protein,the SH2-domain of which binds to phosphotyrosine containing proteins andpromotes signal transduction by causing the association of specificcellular proteins. Cloning of Grb2 is described in Lowenstein et al.,Cell 70:431-442 (1992). IRS-1 is a substrate for the insulin receptor(IR) tyrosine kinase, and binds to the SH2 domain of Grb2 after beingtyrosine phosphorylated. (The interaction of IRS-1 with Grb2 isdescribed in Skolnick et al., EMBO 12:1929-1936 (1993).)

Preferred ligands for this assay have a dissociation constant of lessthan about 1 μM. The present assay employs a synthetic ligand for theSH2-domain that is a phosphotyrosine containing peptide derived from thesequence of IRS-1 (located near the pY896 amino acid). The peptide hasthe sequence KSPGNpYVNIE-CO₂ NH₂.

Streptavidin coated 6.2 μm beads (Spherotech) are employed that contain1×10⁶ binding sites per bead. The beads are incubated with theSH2-domain (Src-homology 2 domain), which has been labelled at aspecific site with biotin during expression (Schatz, P. J. (1993)Bio/Technology 11, 1138-1143).

Beads, ligand, and library compounds are combined, incubated and shakenin 1536 well plates. The samples are then allowed to settle for about 10minutes, and the plates read by a laser scanning confocal microscope.Active compounds cause a decrease in the amount of bound fluorescence.Results are analyzed as described above in Example 1 to determinebinding and/or inhibition constants.

EXAMPLE 3 Screening of compounds for EGF Receptor Kinase Domain binding

A high throughput assay is performed to determine library compounds thatinhibit binding of ligand to the kinase domain of the epidermal growthfactor (EGF). This kinase phosphorylates a number of proteins andpeptides, including the peptide RRKGSTAENAEYLRVA (the "1173" peptide).The tyrosine underlined in the amino acid sequence is phosphorylated bythe kinase.

EGF Receptor Kinase Domain is obtained from Alexis Biochemicals (SanDiego, Calif.). The 1173 peptide is biotinylated and then incubated withstreptavidin coated 6.2 μKm microbeads (Spherotech) to affix the peptideto the beads. Monoclonal antibodies specific for phosphotyrosine areobtained from Sigma (St. Louis, Mo.) or Biogenesis (Sandown, N.H.), andare labelled with Cy5 following the manufacturer's directions(Amersham).

The assay is conducted in 1536 well plates. The EGF kinase domain isincubated in the presence of the 1173 coated microbeads, MgATP, Cy5labelled anti-phosphotyrosine antibody, and library compound in neutralbuffer. Upon phosphorylation of the 1173 peptide by the kinase, thepeptide is bound by the fluorescently labelled antibody, andfluorescence becomes associated with the beads. Inhibitory librarycompound prevents phosphorylation, resulting in a decrease in beadassociated fluorescence. The wells of the plate are scanned by a laserscanning confocal microscope, and the resulting data further analysed asdescribed in Example 1 to determine inhibition and/or binding values.

We claim:
 1. A high throughput assay for rapidly screening a pluralityof compounds to determine the degree of inhibition of a ligand/receptorinteraction or of an enzyme catalyzed reaction by said compounds, or todetermine the degree of binding of said compounds to a target molecule,said assay comprising:(a) a step selected from the group consistingof(1) contacting said compounds to be tested with said ligand and saidreceptor; (2) contacting said compounds to be tested with said enzymeand substrate for said enzyme; and (3) contacting said compounds to betested with said target molecule;wherein inhibition or binding by one ormore of said compounds causes a change in the amount of an opticallydetectable label bound to suspendable cells or solid supports present insaid assay; (b) determining said degree of inhibition or binding by:measuring through use of confocal microscopy amounts of said opticallydetectable signal bound to individual cells or solid supports; comparingsaid amounts with an amount of back-ground signal in said assay that isnot bound to said cells or solid supports; and determining said degreeof inhibition or binding from the difference between said bound andbackground signal,wherein said assay is homogeneous.
 2. The assay ofclaim 1 comprising an assay for enzyme inhibitors.
 3. The assay of claim1 comprising an assay for inhibitors of receptor binding.
 4. The assayof claim 3 wherein said receptor consists of a receptor domain thatbinds to said ligand.
 5. The assay of claim 1 wherein said opticallydetectable label is selected from the group consisting of achemiluminescent label, a fluorescent label, and an enzyme that producesa visible color change.
 6. The assay of claim 1 wherein said ligand isfluorescently labelled.
 7. The assay of claim 1 wherein said librarycompounds are fluorescently labelled.
 8. The assay of claim 2 comprisingan assay wherein enzyme and library compound in solution are incubatedwith fluorescently labelled substrate that is attached to suspendablesolid supports, said incubation resulting in release of fluorescentlylabelled reaction product in the absence of inhibitory compound.
 9. Theassay of claim 2 wherein enzyme and fluorescently labelled substrate areincubated with suspendable solid supports that are coated with a moietythat specifically binds to the reaction product of said enzyme/substratereaction, and wherein said reaction product is fluorescently labelled.10. The assay of claim 1 wherein said receptor is labelled and saidligand is bound to said suspendable solid supports.
 11. The assay ofclaim 1 wherein said confocal microscopy is laser scanning confocalmicroscopy.
 12. The assay of claim 1 wherein said confocal microscopyemploys a Nipkow disc.
 13. The assay of claim 1 wherein said compoundsare contacted with enzyme and substrate, and inhibit by a mechanismselected from the group consisting of binding to said enzyme, binding tosaid substrate, binding to a complex of enzyme and substrate, andbinding to a complex of enzyme and product.
 14. The assay of claim 1wherein prior to step (b) said suspendable cells or solid supports areallowed to settle in containers in which said assay is conducted suchthat more than about 75% of said cells or solid supports are settled inless than about 25% of the volume of said containers.
 15. A highthroughput assay for rapidly screening a plurality of compounds forinhibition of binding of a fluorescently labeled ligand to a targetmolecule contained on suspendable cells or solid supports, said methodcomprising(a) contacting said fluorescently labeled ligand with saidtarget molecule contained on said suspendable cells or suspendable solidsupports in the presence of said compounds; (b) measuring the degree ofinhibition of said binding by determining through use of confocalmicroscopy amounts of fluorescently labeled ligand bound to individualcells or solid supports; (c) comparing said amounts with fluorescence insaid assay that is not associated with said cells or solid supports, and(d) correlating said difference with said degree of inhibition, whereinsaid assay is homogeneous.
 16. The method of claim 15 wherein saidtarget molecule is a receptor or receptor domain.
 17. The method ofclaim 16 wherein said receptor or receptor domain is contained on thesurface of suspended cells.
 18. The method of claim 15 wherein saidtarget molecule is attached to a suspendable solid support.
 19. Themethod of claim 18 wherein said suspendable solid support is less thanabout 50 μm in diameter.
 20. The method of claim 18 wherein saidsuspendable solid support is less than about 10 μm in diameter and morethan about 1 μm in diameter.
 21. The assay of claim 15 wherein prior tostep (b) said suspendable cells or solid supports are allowed to settlein containers in which said assay is conducted such that more than about75% of said cells or solid supports are settled in less than about 25%of the volume of said containers.
 22. The assay of claim 15 wherein saidconfocal microscopy is laser scanning confocal microscopy.
 23. Ahomogeneous assay for screening a library of compounds for inhibition ofbinding between a ligand and a cell surface receptor or receptor domaincomprising:a) adding a liquid suspension of cells expressing said cellsurface receptor or domain to a plurality of containers; b) adding alibrary of compounds to be screened for said inhibition to saidplurality of containers; c) adding a fluorescently labelled ligand forsaid receptor to said containers; d) incubating said cells, saidcompounds, and said fluorescently labelled ligand; e) individuallymeasuring amounts of fluorescence associated with said cells in saidcontainers by confocal microscopy; and f) determining the degree ofinhibition by one or more of said compounds of binding of said ligand tosaid receptor.
 24. The method of claim 23 wherein, following saidincubation and prior to said measurement, said cells are allowed tosettle in said containers such that more than about 75% of said cellsare settled in less than about 25% of the volume of said containers. 25.The method of claim 23 wherein, following said incubation and prior tosaid measurement, said cells are allowed to settle in said containerssuch that more than about 90% of said cells are settled in less thanabout 10% of the volume of said containers.
 26. The method of claim 23wherein said compounds are fluorescently labelled with a moiety thatfluoresces at a wavelength above about 600 nm.
 27. The method of claim23 wherein said containers comprise wells in one or more microtiterplates.
 28. The method of claim 23 wherein said containers compriseabout 1 μl or less of assay solution.
 29. The assay of claim 23 whereinsaid confocal microscopy is laser scanning confocal microscopy.
 30. Ahomogeneous assay for screening a library of compounds for inhibition ofbinding between a ligand and a target molecule attached to a suspendablesolid support comprising:a) adding a liquid suspension of said targetmolecules attached to said suspendable solid supports to a plurality ofcontainers; b) adding a plurality of compounds to be screened for saidinhibition to said plurality of containers; c) adding fluorescentlylabelled ligand to said containers; d) incubating said target moleculesattached to solid supports, said compounds, and said fluorescentlylabelled ligand; e) individually measuring amounts of fluorescenceassociated with said solid supports by confocal microscopy; and f)determining the degree of inhibition by one or more of said compounds ofbinding of said ligand to said target molecule.
 31. The method of claim30 wherein, following said incubation and prior to said measurement,said solid supports are allowed to settle in said containers such thatmore than about 75% of said cells are settled in less than about 25% ofthe volume of said containers.
 32. The method of claim 30 wherein,following said incubation and prior to said measurement, said solidsupports are allowed to settle in said containers such that more thanabout 90% of said cells are settled in less than about 10% of the volumeof said containers.
 33. The method of claim 30 wherein said compoundsare fluorescently labelled with a moiety that fluoresces at above about600 nm.
 34. The method of claim 30 wherein said containers comprisewells in one or more microtiter plates.
 35. The method of claim 30wherein said containers comprise about 1 μl or less of assay solution.36. The assay of claim 30 wherein said confocal microscopy is laserscanning confocal microscopy.
 37. A homogeneous assay for screening alibrary of compounds for inhibition of binding between a ligand and atarget moiety attached to an insuspendable solid support or cellscomprising:a) adding a plurality of compounds to be screened for saidinhibition to containers comprising said insuspendable solid support orcells; b) adding fluorescently labelled ligand to said containers; c)incubating said target moiety attached to said insuspendable solidsupport or cells, said compounds, and said fluorescently labelledligand; d) measuring the amount of fluorescence associated with saidsolid support or cells by confocal microscopy; and e) determining thedegree of inhibition by one or more of said compounds of binding of saidligand to said target moiety.
 38. The assay of claim 37 wherein saidcontainers comprise wells of a microtiter plate, and said insuspendablesolid support comprises the bottom of wells in said plate.